Custom antibody development

We offer world-leading antibody development platforms based on a proprietary RabMAb® rabbit monoclonal platform, a phase display platform (AxioMx), and a next-generation sequencing platform (NGS-RabMAb®)

The antibody has been negatively preadsorbed using a non-phosphopeptide corresponding to the site of phosphorylation to remove antibody that is reactive with non-phosphorylated PDGFR alpha. The final product is generated by affinity chromatography using a PDGFR alpha derived peptide phosphorylated at tyrosine 754.

Applications

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application

Abreviews

Notes

ICC/IF

Use at an assay dependent concentration. 1/50 (see Abreview)

IHC-P

1/20.

WB

Use a concentration of 0.35 - 1 µg/ml. Detects a band of approximately 170-185 kDa.

Target

Function

Receptor that binds both PDGFA and PDGFB and has a tyrosine-protein kinase activity.

Tissue specificity

Expressed in primary and metastatic colon tumors and in normal colon tissue. Tumors may express a different isoform to that found in normal tissue.

Involvement in disease

Note=A chromosomal aberration involving PDGFRA is found in some cases of hypereosinophilic syndrome. Interstitial chromosomal deletion del(4)(q12q12) causes the fusion of FIP1L1 and PDGFRA (FIP1L1-PDGFRA).

Anti-PDGF Receptor alpha (phospho Y754) antibody images

Peptide Competition: Extracts prepared from NIH3T3 cells left unstimulated (1) and stimulated with PDGF (2-5) were resolved by SDS-PAGE on a 10% polyacrylamide gel and transferred to PVDF. Membranes were blocked with a 5% BSA-TBST buffer overnight at 4°C, then were incubated with 0.50 µg/mL ab5460 antibody for two hours at room temperature in a 1% BSA-TBST buffer, following prior incubation with: no peptide (1, 2), the non-phosphopeptide corresponding to the immunogen (3), a generic phosphotyrosine containing peptide (4), or, the phosphopeptide immunogen (5). After washing, membranes were incubated with goat F(ab’)2 anti-rabbit IgG alkaline phosphatase and bands were detected using the Tropix WesternStar method. The data show that only the peptide corresponding to ab5460 blocks the antibody signal, thereby demonstrating the specificity of the antibody. Peptide Competition: Extracts prepared from NIH3T3 cells left unstimulated (1) and stimulated with PDGF

ab5460 staining PDGF Receptor alpha (phospho Y754) in murine brain tissue by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections). Tissue was fixed with paraformaldehyde and a heat mediated antigen retrieval step was performed using citric acid pH 6.1. Samples were then permeabilized using 0.3% H2O2, then blocked with 0.5% BSA for 20 minutes at room temperature, followed by incubation with the primary antibody at a 1/50 dilution for 1 hour. An undiluted HRP-conjugated Goat anti-rabbit/mouse polyclonal was used as secondary antibody. Nuclei staining with Haematoxillin (blue) and PDGF Receptor alpha staining with DAB (brown). PDGF Receptor alpha staining in the membrane of astrocytes.

ab5460 staining PDGF Receptor alpha (phospho Y754) in human glioblastoma multiforme brain tissue by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections). Tissue was fixed with paraformaldehyde and a heat mediated antigen retrieval step was performed using citric acid pH 6.1. Samples were then permeabilized using 0.3% H2O2, then blocked with 0.5% BSA for 20 minutes at room temperature, followed by incubation with the primary antibody at a 1/50 dilution for 1 hour. An undiluted HRP-conjugated Goat anti-rabbit/mouse polyclonal was used as secondary antibody. Nuclei staining with Haematoxillin (blue) and PDGF Receptor alpha staining with DAB (brown). PDGF Receptor alpha staining in the membrane of astrocytes.

I am sorry about the issues you are having with ab5460 in western blot. As we discussed I am sending you a new vial of the antibody to see if that helps resolves the situation. Your new order number i...